Summary This is a proposal for Renewal of R01 CA168611. Pancreatic ductal adenocarcinoma (PDA) is characterized by immune-suppressive inflammation enables PDA progression. In the past funding period we showed that pattern recognition receptor (PRR) signaling in tumor-associated macrophages (TAMs) drives the tolerogenic innate immune program in PDA and plays a central role in suppressive T cell differentiation. However, the regulatory mechanism governing PRR-driven macrophage programming and the ?upstream? signals that exert global control over immune tolerance in PDA are unknown. Our current proposal will bridge this knowledge gap and provide a novel target for immunotherapy. Receptor-interacting protein 1 (RIP1) is a kinase that modulates NF-?B-mediated inflammation, MAP Kinase signaling, promotes apoptosis, and is required for organized necrosis. We postulate that RIP1 is a master upstream driver of PRR-mediated tolerogenic macrophage programming and is necessary for macrophage-mediated T cell suppression in PDA. Our proposal is divided into 3 new Aims which build on our previous work: Aim 1 will investigate whether RIP1 inhibition is protective in diverse murine models of PDA. We will assess the efficacy of targeting RIP1 using a novel small molecule RIP1 inhibitor and in mice with genetic RIP1 deficiency. We will also test our primary hypothesis that RIP1-mediated inflammatory signaling leads to the expansion of immune-suppressive TAMs which, in turn, generate tolerogenic T cells leading to tumor- permissive anergy. Further, we will evaluate our translational hypothesis that targeting RIP1 will have synergistic efficacy with checkpoint-receptor directed immunotherapeutic regimens. We will define the biochemical mechanisms of RIP1-mediated immune-suppression by evaluating our hypothesis that RIP1 induces immune- suppressive PRR signaling and elaboration of immunomodulatory chemokines leading to tolerogenic macrophage programming. In Aim 2, to extend our findings towards clinical translation, we will test our hypothesis that RIP1 signaling triggers PRR signaling, intra-tumoral immune-tolerance, and enables tumor growth using an innovative human 3D organotypic cancer model which we validated both in terms of its fidelity to human PDA and as a platform for evaluating immune-based therapies. In Aim 3 we will test whether RIP1 inhibition, alone or in combination with checkpoint-based immunotherapies, is safe and efficacious in advanced PDA patients within the context of Phase 1 clinical trial (NCT03681951). We will prospectively assess changes in anti-tumor immunity and PRR signaling following RIP1 inhibition using innovative protein- and RNA-based methodologies. In summary, our proposed work will detail the mechanism of RIP1-mediated immune-suppression in PDA in sophisticated mouse models and in a human 3D organotypic culture system and will investigate the efficacy of targeting RIP1 in reversing immune-suppression in a cutting-edge clinical trial.